Neuronal synapses undergo rapid alternations in morphology and protein distribution to process and integrate information. Many of the processes occur on a millisecond time scale in a confined space. To visualize these events, I have developed two novel techniques in electron microscopy. One technique stimulates neurons using optogenetics and captures the subsequent morphological changes at millisecond temporal resolution. The second technique couples super-resolution imaging and electron microscopy to identify the locations of proteins within ultrastructure. Using these techniques, we have discovered a novel endocytic pathway that retrieves synaptic vesicle membrane within 100 ms after fusion. We further demonstrated that the internalized membrane is delivered to endosomes and that clathrin regenerates synaptic vesicles from the endosomes. Currently, we are studying lipid regulation during synaptic vesicle recycling. Our preliminary results indicate that synaptic endosomes have unique lipid constituents. However, several key tools are missing and will be required to study the regulation of lipid constituents in these transient structures at synaptic terminals: 1) a method to visualize lipid dynamics at sub-second temporal resolution; 2) an approach that allows spatially and temporally resolved lipidomic analysis to reveal the lipid constituents of transient structures at synapses, and 3) a method to manipulate lipid composition locally at endosomes. In this proposal, we aim to develop these tools and leverage them to study lipid regulation at synapses. The proposed study will provide insights into the normal synaptic regulation at unprecedented spatial and temporal resolution, and will eventually help elucidate how synaptic function is impaired in disease conditions - a wide range of neurological disorders have been linked to defects in synaptic membrane trafficking. In addition, the developed techniques can be adapted by cell biologists in all disciplines to study membrane remodeling with a previously unattainable degree of spatial and temporal precision.